The present invention relates to a method for determining endotoxin concentrations in samples and, if present, potentially interfering factors.
The inventors have already described such a method based on the addition of defined amounts of endotoxin to a sample and computational evaluation of the reaction kinetics; cf. Arzneimittel-Forschung 33 (I), 5, 681-687 (1983) Editio Cantor. The method is based on the known fact that the lysate of the amoebocytes from the hemolymph of the horseshoe crab (Limulus polyphemus; Tachypleus tridentatus et al.) forms a gel in the presence of endotoxins. This gel-forming reaction proceeds via an enzyme chain of which not all steps have been elucidated. This basic principle for the detection of endotoxin is used, for example, in the semi-quantitative tube gelation test (clot test), the turbidimetric endpoint or two-point determination and various methods for assaying the Limulus Amoeboctye Lysate-Endotoxin (LAL-ET) reaction kinetics.
In the conventional quantitative methods it is required to investigate several sample dilutions because of the narrowly limited range of measurement. In addition thereto, erroneous determinations may occur, more particularly in testing substances which will interfere with the detection reaction. So far it has not been possible to obtain insight into the behavior of a sample as to type and extent of said interference and, at the same time, to determine the endotoxin content thereof.
According to inventor's aforementioned publication, in a very meticulous and expensive procedure increasing amounts of a standardized endotoxin were added to preparations of the sample to be tested, and the reaction kinetics were determined by means of a quantitative turbidimetric LAL microtiter test. Typical examples of such reaction kinetics have been shown in FIG. 1 of the publication. The maximum increase in the optical density per minute vs. the logarithm of the endotoxin concentration provides an endotoxin standard curve which is linear throughout an extremely large range. Such a typical endotoxin standard curve comprising a range of from 8 to 4,000 pg/ml is shown in FIG. 2 of the publication.
Furthermore, reaction profiles were determined with constant spikes of endotoxin by assaying sample dilutions comprising constant endotoxin spikes. Depending on the tested sample these reaction profiles resulted in an inhibition, enhancement or in the absence of interfering factors. Such typical reaction profiles are shown in FIG. 3 of the publication.
Frequently, the determination of unknown amounts of endotoxins is significantly affected by interfering factors. Thus, reliable results so far have only been obtainable if, upon investigation and evaluation of the reaction kinetics, an internal standardization has been determined by means of an elaborate mathematical computation in accordance with a non-linear model.